The Nerve Growth Factor (NGF) was originally discovered in mouse sarcoma tumors (Levi-Montalcini R. et al., J. Exp. Zool. 116:321, 1951) and was then purified to homogeneity from submandibular salivary glands of male mice (Varon S. et al., Biochemistry 6:2202, 1967) and from snake venom (Angeletti R. H., Proc. Natl. Acad. Sci. USA 65:668, 1970). Many other relatively rich sources of NGF have also been reported, including guinea pig prostate (Harper G. P. et al., Nature 279:160, 1979) and human placenta (Goldstein L. D. et al., Neurochem. Res. 3:175, 1978; Walker P. et al., Life Science 26:195, 1980; Calissano P. et al., Hormonal Prot. Peptides, XII:2, 1984). Small quantities of NGF have been reported to be present in other tissues including the mammalian central nervous system (Varon S., Discussions in Neuroscience, vol. II, n.degree. 3, 1985; Hefti F. et al., Neuroscience 14:55, 1985). The physiological relationship between these potential sources of NGF and the apparent action sites is not very clear, but it is generally supposed that NGF is secreted by various peripheral tissues requiring innervation by those cells which respond to NGF.
The sequence and cloning of NGF obtained from submandibular glands of male mice were also carried out (Scott J. et al., Nature 302 538, 1983; Ulrich A. et al., Nature 303:821, 1983). The human .beta.NGF gene has also been successfully isolated and cloned (Ulrich A. et al., Nature 303:821, 1983; European Patent n.degree. 0121388).
NGF obtained from submandibular glands of mice was the type most completely characterized. NGF from mouse glands acts as a 7S proteic complex (molecular weight about 140,000 daltons) of three different subunits (.alpha., .beta., .gamma.) including Zn.sup.+. The activity of NGF is exclusively associated with the subunit .beta. (known as 2.5S NGF), a basic dimeric protein with a molecular weight of about 25,300 daltons (showing a molecular weight of about 12,650 daltons on electrophoresis with gel at high concentration of SDS and after its reduction with .beta.-mercaptoethanol at 100.degree. C. for 5 minutes), the isoelectric point of which is approximately 9.3. The amino acid sequences of .beta.-NGF from submandibular glands of male mice and human provenience have been reported (Scott J. et al., Nature 302:538, 1983; Ulrich A. et al., Nature 303:821, 1983)
NGF from mouse submandibular gland was used for most of the studies on the activity of NGF in vivo and in vitro The range of biological activity in vitro of NGF has been determined both on primary neuronal cells and on clonal cells in cultures The primary neuronal cells reported as responding to NGF in vitro include fetal sensorial neurons (embryonic day 8-12) in dorsal root ganglia, noradrenergic fetal neurons in the sympathetic ganglia, cholinergic fetal neurons in the septum and adrenal chromaffin cells in development. While sensorial and sympathetic neurons depend on NGF for survival and development, cholinergic neurons do not seem to require NGF for survival, but only for their differentiation, that is to say, the expression of characteristic phenotypic traits bound to the neurotransmitter. The addition of NGF to the adrenal chromaffin cells (cells derived the neural crest) in the initial stage of their development causes the expression of neuronal phenotypes. The clonal cells reported as responding to NGF in vitro include chromaffin suprarenal cells derived from tumors of the neuronal crest known as pheochromocytoma cells (PC12) and human neuroblastoma cells. After treatment with NGF these cells switch from a highly proliferous form of behaviour to a postmitotic neuronal state. Recently it has become possible to measure both NGF and its mRNA in several rat brain regions. A striking correlation of NGF level with the distribution of magnocellular cholinergic neurons has been found. Relatively high NGF levels in the range of those observed in peripheral sympathetic target tissues were found both in the regions innervated by magnocellular cholinergic neurons and in the regions containing their cell bodies, i.e. the hippocampus. The brain regions not related to the magnocellular cholinergic system contain considerably lower levels of NGF. The magnocellular cholinergic neurons of the basal forebrain project topologically to the neocortex, hippocampus and olfactory. The learning ability and the memory of the rodents have been associated with an age-dependent decline of cholinergic function in the forebrain and recent evidence indicates that the cholinergic neurons in the nucleus basalis magnocellularis, the septal-diagonal band area and the striatum undergo age-dependent atrophy. The spatial memory can be partly restored by intraventricular injection of NGF. This relation between the integrity of the basal forebrain cholinergic system and cognitive functions may also be valid for human beings. One of the main neuropathologic features of Alzheimer's disease is the drastic loss of magnocellular cholinergic neurons, although other transmitter systems undergo several changes. A correlation has been proposed between the extent of damage to the cholinergic system and the severity of the mental deficits.
The possible links between NGF and the pathophysiology and potential therapy of Alzheimer's disease have thus moved into the range of realistic consideration.
The involvement of the cholinergic system in the clinical manifestations of Alzheimer's disease is also supported by various experimental observations. For instance, in rats the interruption of the ascending cholinergic projections from the basal forebrain nuclei results in a marked reduction of memory and learning ability. These learning and memory deficiencies can be improved by injecting NGF into the hippocampus. The available pathophysiological information from Alzheimer patients, and the complementary information from animal experiments, open up interesting possibilities for the elucidation of the pathophysiological causes of Alzheimer's disease and potential new therapeutic approaches.
The availability of cDNA probes for human NGF, the possibility of producing human NGF by biotechnological methods, the consequent production of specific antibodies against human NGF, and the development of a specific enzyme immunoassay are all prerequisites for an experimental approach to the question as to whether Alzheimer's disease is actually associated with a deficit in the production of human NGF. If such a deficit were found, it would also be necessary to postulate that as well as a reduced production of NGF, there is also a reduced production of other, unknown neurotrophic factors acting on populations of neurons, which are also affected by Alzheimer's disease, but which are not responsive to NGF.
With respect to the therapeutic consequences, the benefits of NGF administration on learning deficits after experimental lesions of the cholinergic system suggest that, whatever the cause of the damage of the cholinergic neurons, an increased availability of NGF for these neurons either by exogenous application or by stimulation of endogenous production, could be substantial.
Since the production of human NGF by biotechnological methods is possible, potential immunological pitfalls of a therapy with non-human NGF could be eliminated.
For many years NGF research has been primarily based on NGF purified from the male mouse salivary gland and the antibodies produced against it. At a relatively early stage of NGF research it became apparent that the injection of anti-mouse NGF antibodies into chick embryos did not result in the same extensive destruction of the sympathetic nervous system as observed after antibody injections into newborn mice and rats. Since chick sympathetic and sensory neurons respond to mouse NGF in vivo and in vitro in a way similar to the corresponding mouse neurons, it was reasonable to conclude that the domain of the NGF molecule responsible for its biological activity must have been preserved, whereas other domains had changed during evolution. This assumption was further substantiated when bovine NGF was purified from bovine seminal plasma, and a detailed and comprehensive comparison between the biological activity of the mouse and bovine NGF became possible. These experiments demonstrated that the biological activity of mouse and bovine NGF were identical, although immunological cross-reactivity was very limited. The molecular cloning of mouse, human, bovine, and chick NGF, together with the amino acid sequence analysis of mouse NGF, has allowed comparison of the conserved and unconserved domains of these molecules and their relationship to biological activity and antigenicity. The overall conservation of NGF during evolution is remarkably high. Of the 118 amino acids of mature mouse .beta.-NGF, only 16 amino acids were changed in bovine NGF, 19 in chick NGF, and 11 in human NGF. As was expected from previous observations, in the reduction of the three S--S bridges of mouse NGF one can obtain a complete loss of biological activity. The apparent discrepancy between the overall high conservation of the amino acid sequence and the poor immunological cross-reactivity is due to the fact that the amino acid changes between species are located in clusters. Hydropathy plots demonstrated that the changes are virtually exclusively located in the hydrophilic domains expected to be potential antigenic determinants. One single hydrophilic region has been shown to be strictly conserved in the NGF molecules of all species investigated so far. This conserved domain lends itself to future analysis by site-directed mutagenisis and by antibodies directed against synthetic peptides corresponding to this region.
The presence of three disulfide bonds in the correct conformation in the monomeric subunit of .beta.-NGF represents a characteristic for this protein in terms of biological activity and immunogenicity.
Rigorous characterization between the native protein and DNA-derived product, both in the active form, is essential. Particular attention should be given to using a wide range of analytical techniques exploiting different physicochemical properties of the molecule; for instance, size, charge, isoelectric point, amino acid composition and hydrophobicity. It may be desirable to include suitable tests to establish that the product has the desired conformational structure and state of aggregation. Examples of techniques suitable for such purposes are: polyacrylamide gel electrophoresis; isoelectric focusing; size exclusion, reversed phase, ion exchange, hydrophobic interaction and affinity chromatography; peptide sequence mapping; amino acid analysis; light scattering; UV spectroscopy; circular dichroism, and other spectroscopic techniques. Additional characterization of the product using, for example, electron microscopy or immunochemical techniques may provide valuable information. Biological and immunological characterization should include as wide a range of techniques as is possible, appropriate to the anticipated biological activity. The determination of the specific activity of highly purified material will be of particular value (units of activity/weight of product).
While evaluating the potential pharmaceutical application of the human NGF (.beta. subunit) previously discussed and the Problems correlated with the DNA-derived first generation products, obtained in E. coli, the present inventors developed a scheme for the purification of NGF (.beta. subunit) from human placenta, potentially useful for large-scale applications. This material is characterized in terms of chemical, immunochemical and biological characteristics using the techniques and the reagents suitable for such purposes. Obviously, this purification scheme or only a single step can be applied to the purification of human NGF, obtained by means of recombinated DNA technology, which shows the same chemical, immunochemical and biological characteristics of NGF purified from human placenta.
Pharmaceutical compositions in accordance with the invention include .beta.-NGF purified from human tissues, a .beta.-NGF analog, biologically active fragments of .beta.-NGF or of analog .beta.-NGF, or nontoxic salts thereof, dispersed in a Pharmaceutically acceptable liquid or in a solid carrier. Such pharmaceutical compositions can be used in clinical medicine, both human and veterinary, in acute or chronic administrations for diagnostic or therapeutic purposes without problems of immunogenicity.
The present invention takes advantages:
of the different isoelectric points of the native 7S complex and the .beta. subunit for the isolation and purification of .beta.-NGF using cation exchange resins and PA1 of the cross-reactivity of human NGF (.beta. subunit) with the polyclonal antibodies produced against mouse NGF (.beta. subunit). PA1 a) the molecular weight of the purified material, evaluated by SDS gel eletrophoresis, was approximately 24.3-25.3 Kdaltons (FIG. 1); PA1 b) the isoelectric point of this material was approximately 9.3-9.8; PA1 c) by means of western blot technique, using a concentration (0.5 .mu.g/ml) of affinity purified polyclonal antibodies produced against mouse NGF, the hNGF (.beta. subunit) from human placenta showed a cross-reactivity against this reagent (FIG. 2).